Anti viral therapy

ABSTRACT

The present invention provides a method of identifying host cell molecules which may be modulated to inhibit viral replication and method of testing antiviral compounds. In addition, the invention provides compositions, methods and medicaments for treating viral infections and/or diseases or conditions caused or contributed to by viruses.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national phase application ofInternational Application Serial No. PCT/GB2010/000623, filed Mar. 30,2010, which claims priority to Great Britain Application No. 0905485.9,filed Mar. 30, 2009, the entire contents of each of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides a method of identifying host cellmolecules which may be modulated to inhibit viral replication and methodof testing antiviral compounds. In addition, the invention providescompositions, methods and medicaments for treating viral infectionsand/or diseases or conditions caused or contributed to by viruses.

BACKGROUND OF THE INVENTION

Antivirals currently on the market use small molecules as the activepharmaceutical ingredient and tend to be specific to certain (types of)viruses. However, through the incorporation of random mutations etc.,viruses can easily evade the effects of antivirals targeting viralproteins, or parts of proteins.

MicroRNAs (miRNAs) are small RNAs (22 nt) that regulate eukaryotic geneexpression by binding to specific messenger RNA transcripts, causing themRNAs to be degraded or causing their translation to be repressed(Bartel 2004). MicroRNAs are encoded in the genomes of animals, plantsand viruses; these genes are transcribed by RNA polymerase II as part oflarger fold-back transcripts (primary miRNAs), which are processed inthe nucleus by Drosha family members to form short stem-loops(pre-miRNAs), and then exported to the cytoplasm for processing by aDicer family member to form the mature miRNA. It is estimated thatmiRNAs comprise 1% of genes in animals and may target up to 30% of genesin humans (Lewis, Burge et al. 2005). A given microRNA can potentiallytarget hundreds of genes and by modulating a whole network of genetargets, can exert dramatic effects on various cellular processes(Giraldez, Mishima et al. 2006). The mechanism of microRNA function(generally down-regulation of host proteins) is distinct, but perhapscomplementary, to other regulatory molecules (e.g. transcriptionfactors). MicroRNAs have been shown to play key roles in cellularproliferation, differentiation, development and neuronal function;specific miRNAs also play a role in cancer formation, cardiovascular andmetabolic diseases, and, more recently, viral infection.

MicroRNAs are an important component of viral-host interactions and havebeen shown to influence the outcome of viral infections, reviewed in(Ghosh, Mallick et al. 2008; Gottwein and Cullen 2008; Kumar 2008).Recent studies have demonstrated that specific host microRNAs are up- ordown regulated upon infection with a particular virus and that some ofthese host microRNAs have target sites against specific viruses. Thishas led to the suggestion that microRNAs could mediate anti-viraldefense; for example, mir-32 was shown to limit the replication ofprimate foamy virus (PFV) in human cells by targeting regions in the PFVgenome (Lecellier, Dunoyer et al. 2005). In another study, mir-24 andmir-93 were shown to target vesicular stomatitis virus, leading todecreased replication of the virus in mice (Otsuka, Jing et al. 2007).However, rather than being “anti-viral”, the host microRNAs that targetviruses may in fact be exploited by the viruses for persistence. From anevolutionary point of view, if the host miRNA target sites weredisadvantageous to the virus, the virus could readily evolve toeliminate these sites (requiring only a single mutation) (Mahajan, Drakeet al. 2008). For example, it was shown that host microRNAs (mir-28,mir-125b, mir-150, mir-223 and mir-382) down regulate HIV mRNA and maybe used by the virus to avoid being eliminated by the immune system(Huang, Wang et al. 2007). Furthermore, it is known that the hostmicroRNA—mir-122, can actually be exploited by the virus to upregulateviral genes (by unknown mechanisms) (Jopling, Norman et al. 2006). Thework listed above demonstrates that human or mouse microRNAs can play apro- or anti-viral function by interacting with viral sequences;however, antiviral therapies based on microRNAs that specificallyinteract with viral genomes possess a number of disadvantages: 1) theviruses can mutate/evolve to escape the microRNA-target interactions 2)the identified microRNAs would be limited to function against the viruswith the target site, rather than holding broad anti-viral potential.

More recently, it has been shown that cellular microRNAs that areinduced or down regulated upon viral infection can also modulate hostgenes, which are co-factors for viral infection. For example, the miRNAcluster mir-17/92 was shown to be decreased upon HIV-1 infection and wasshown with knockdown experiments to effect HIV-1 replication; thismicroRNA targets histone acetylase protein PCAF, which is a co-factorfor the viral Tat protein (Triboulet, Mari et al. 2007).

SUMMARY OF THE INVENTION

The present invention is based on the finding that micro RNA (miRNA)molecules may be exploited as a means of treating viral infections. Morespecifically, the inventors have discovered that by modulating theexpression of one or more host cell miRNA molecules, it is possible toinhibit the replication and/or propagation of one or more viral speciesin a host cell.

The inventors have observed that when over expressed, certain host cellmiRNA molecules may exhibit antiviral effects. When these same host cellmiRNA molecules are inhibited, viral propagation and/or replication inthe host cell may increase. In other instances, certain host cell miRNAmolecules are pro-viral and thus by inhibiting these host cell miRNAs,it is also possible to achieve an antiviral effect.

It should be understood that the definitions of the various terms usedin this specification apply to all aspects and embodiments of thisinvention.

In a first aspect, the present invention provides a method of screeningfor, or identifying, host miRNA molecules which modulate viralpropagation and/or replication, said method comprising the steps of:

(a) introducing a host cell miRNA modulating compound into a host cell;

(b) contacting the host cell with one or more viruses under conditionsto permit infection of the cell with said virus(es);

(c) identifying any modulation of viral propagation and/or replicationin said host cell;

wherein modulated viral propagation and/or replication indicates thatthe host cell miRNA modulating compound modulates a host cell miRNAwhich may modulate viral propagation and/or replication.

Where step (b) involves contacting host cells with two or more virusesof different species, it may be possible to identify host miRNAmolecules which, when modulated, result in multi-species orbroad-spectrum, antiviral activity. That is to say, rather than simplyinhibiting the propagation and/or replication of a single viral species,modulation of a host cell miRNA identified using the methods describedherein, may inhibit the propagation and/or replication of a number ofdifferent (i.e. two or more) viral species. It should be understood thatthe term “multi-species” may encompass two or more different viralspecies.

Without wishing to be bound by theory, the inventors hypothesise thatthe host miRNA molecules identified by the method described hereintarget host cell genes rather than viral genes and as such, viruses areunable to mutate or evolve to escape the effects of modulating thesemiRNA molecules.

It should be understood that the term “modulation” encompasses bothincreased and/or decreased (or over- or under-) expression as well asup- or down-regulation events. Furthermore, the term “modulation” coversany increase and/or decrease in function and/or activity. For example,“host cell miRNA modulating compounds” may be compounds which increaseand/or decrease the expression, function and/or activity of certain hostcell miRNA molecules. In other embodiments, compounds which modulate theactivity and/or function of a host cell miRNA molecule, may be thosethat mimic or inhibit the effect of said host cell miRNA molecules. Thephrase “modulation of viral propagation and/or replication” encompassesany increase and/or decrease in levels of viral propagation and/orreplication. Similarly, “modulation of host cell miRNA molecules”encompasses increased or decreased expression, function and/or activityof host cell miRNA molecules.

Modulation of host miRNA molecules and/or viral propagation/replication,may be detected relative to the levels of host cell miRNA moleculeexpression or viral propagation and/or replication occurring in cellsinto which host cell miRNA modulating compounds have not beenintroduced. Additionally or alternatively, a control molecule may beintroduced into the cell instead of host cell miRNA modulatingcompounds. Control molecules may take the form of molecules which do notmimic or inhibit host cell miRNA molecules.

Where the methods provided by the first aspect of this invention do notinvolve the introduction of host cell miRNA modulating compounds orutilise control molecules, these methods may be referred to asreference, standard or control methods. In all cases, the resultsobtained from the methods provided by the first aspect of this inventionmay be compared to the results obtained from reference, standard orcontrol methods. Other control, reference or standard methods may lackthe step of contacting cells with one or more viruses (step (b)).

One of skill in this field will be familiar with the term “miRNA” whichencompasses single-stranded RNA molecules which regulate geneexpression. miRNA molecules may be between 10 and 50 nucleotides inlength, preferably 15-40, more preferably 16-30 and even more preferably17-25 nucleotides in length. Typically, miRNA molecules may be between19 and 26 nucleotides in length. Further information concerning miRNAmolecules may be found in, Lagos-Quintana, M., R. Rauhut, W. Lendeckel,and T. Tuschl. “The C. elegans heterochronic gene lin-4 encodes smallRNAs with antisense complementarity to lin-14”. Cell 75 (5): 843-54.

In the context of this invention the terms “virus”, “viruses” or “viral”are intended to encompass all types of viruses including, for example,DNA and/or RNA viruses. As such, the methods provided by this inventionmay identify host miRNA molecules which, when modulated, inhibit thepropagation and/or replication of a variety of different viruses ofdifferent species. In other words modulation of host cell miRNAidentified by the methods provided by the first aspect of this inventionmay result in multi-species or broad spectrum antiviral activity—i.e.antiviral activity which is not specific to just one viral species butto many (i.e. two or more) viral species.

Cells may be contacted with one or more viruses selected from the groupconsisting of murine cytomegalovirus (MCMV), mouse gammaherpese virus,herpes simplex virus—type I, Semliki-forest virus and humancytomegalovirus (HCMV). One of skill in this field will understand thenumber of viruses which may be used, (DNA and/or RNA), is large and thatthe exact choice, may depend on the particular type of cell used.

The steps involved in introducing a host cell miRNA modulating compoundinto a cell are well known and may involve, for example, the use oftransfection protocols or vectors (for example eukaryotic geneexpression vectors) such as transcription cassettes, plasmids or viralvectors.

Typically, transfection protocols, including reverse transfectionprotocols, utilise conditions rendering cell membranes permeable tocompounds such as nucleic acids. By way of example, it may be possibleto transfect (or reverse transfect) host miRNA mimic and/or inhibitormolecules into cells using electroporation, heat shock and/or compoundssuch as calcium phosphate or lipid-based reagents.

Additionally, or alternatively, the host miRNA modulating compound maybe introduced into the cell by means of a gene gun. In such cases, thenucleic acid to be introduced may be associated with or otherwiseconjugated to a particle which can be delivered directly to the cell.

In one embodiment, the host miRNA modulating compound may be introducedinto a host cell in the form of a vector. This is particularly usefulwhere the host cell modulating compound takes the form of a nucleic acid(RNA or DNA) molecule. In this case, the term “vector” may encompassplasmids, viral genomes, or other forms of expression cassette suitablefor introducing nucleic acid sequences to a cell. Further information onsuitable vectors may be found in Sambrook J., Fritsch E., Maniatis T.,Molecular cloning: a laboratory manual, Cold Harbor Spring LaboratoryPress, second edition, 1989.

The term “host cell” encompasses cells capable of supporting viralreplication and as such includes mammalian, such as for example, rodent(mouse, rat, rabbit, guinea pig and the like) or human cells. Plantand/or insect cells are also to be considered as “host cells”. In thisregard, it is to be understood that any type of mammalian, plant orinsect cell may be suitable for use in these methods. In particular,human cells such as fibroblasts, for example MRC-5 fibroblasts may beuseful. In other embodiments, murine cells such as, for example, NIH-3T3cells may be used. Again, it should be understood that the particularchoice of cell may influence the choice of virus(es) to be contactedwith the cell. For example, where the cell is a human cell, step (b)will likely utilise viral species capable of infecting andpropagating/replicating in human cells. Similarly, if the cell is aplant or insect cell, the choice of viral species will likely includespecies capable of infecting and propagating/replicating in plant orinsect cells.

Host cell miRNA modulating compounds may replicate or mimic the sequenceof a host cell miRNA molecule—such compounds are referred to hereinafteras “mimic” miRNA molecules. Mimic miRNA molecules may be exploited as ameans of increasing or upregulating the expression, activity and/orfunction of a particular host cell miRNA. By way of example, the cellmay be contacted or (reverse) transfected with an miRNA molecule whichmimics a host cell miRNA to be up-regulated or over-expressed. In thisway, the normal miRNA expression profile of the host cell issupplemented with the mimic miRNA molecule. In one embodiment, the mimicmiRNA molecules comprise nucleic acid (DNA or RNA) and may themselves bemiRNA molecules.

In other embodiments, the host cell miRNA modulating compounds mayinhibit or down-regulate the expression of a particular host cell miRNA.Such compounds may comprise nucleic acid for example, oligonucleotidesequences, specifically designed to inhibit the expression of one ormore host cell miRNA sequences—compounds of this type will be referredto hereinafter as “inhibitor compounds”. Suitable inhibitor compoundsmay include, for example, DNA or RNA oligonucleotides, preferablyantisense oligonucleotides. Such siRNA oligonucleotides may take theform of single or double-stranded RNA molecules which have been modifiedin some way (for example by chemical modification) to be nucleaseresistant. In order to decrease or down-regulate the expression of aparticular host cell miRNA, or to block the activity of the host cellmicroRNA, the host cell may be contacted or transfected with any of theabovementioned inhibitor compounds. By analysing native or wild-typehost cell miRNA sequences—such as, for example, those described hereinas SEQ ID NOS: 22-26, and with the aid of algorithms such as BIOPREDsi,one of skill in the art could easily determine or computationallypredict nucleic acid sequences that have an optimal knockdown effect forthese genes (see for example: http://www.biopredsi.org/start.html).Accordingly, the skilled man may generate and test an array or libraryof different oligonucleotides to determine whether or not they arecapable of modulating the expression, function and/or activity ofcertain host cell miRNA molecules.

To identify any modulation of viral replication and/or propagation itmay possible to modify the viruses to include some form of reporterelement. For example, the viruses may be modified to include afluorescent or luciferase reporter moiety. Where two or more viruses areto be added to a cell, each virus may be modified to include a differentreporter moieties. The expression of such moieties may easily bedetected using, for example, optical plate readers and the like. Inother embodiments, plaque, complement, antibody, haemolytic and/orhemagglutination assays may be used to detected modulation of viralpropagation and/or replication in a host cell. In all cases the amountor number of fluorescent or luciferase moiety, plaques, hamolysis, celllysis and/or haemagglutination detected, correlates with modulated viralpropagation and/or replication.

As stated, modulated viral propagation and/or replication may easily bedetected by comparing the results obtained from the method provided bythe first aspect of this invention with the results obtained fromcontrol, standard or reference method in which no host cell miRNAmodulating compound has been introduced. In one embodiment, 0.1-3×increases or decreases in the levels of propogation and/or replicationrelative to the level of viral propagation and/or replication observedin a control, standard or reference method, may be taken to indicatemodulated viral propagation and/or replication. Typically 1.5× increasesor decreases in the levels of propogation and/or replication relative tothe level of viral propagation and/or replication observed in a control,standard or reference method, may be taken to indicate modulated viralpropagation and/or replication.

In a further aspect, the present invention provides a means of testingpotential antiviral compounds or drugs. For example, miRNA modulatingcompounds may be tested to determine whether or not they can be used asantivirals. Such experiments may be conducted in cell based systems, forexample in vitro, or in vivo, for example in animals such as rodents. Byway of example, a host cell miRNA modulating compound (for example amiRNA mimic or inhibitor compound) may be introduced into a cell or intoan animal. Thereafter, the host cell or animal may be contacted with oneor more viruses and any modulation of viral propogation and/orreplication may be detected as described above. Those miRNA modulatingcompounds identified as inhibiting viral propogation and/or replicationmay be used in methods, as compounds or in the manufacture ofmedicaments for treating viral infections and/or diseases and/orconditions. Host miRNA sequences are often conserved and miRNA compoundswhich modulate viral propagation and/or replication in, for example,murine systems (including live mice or the like) may be further testedin human systems (for example in human cell lines) and with humanviruses. In certain embodiments, cholesterol modified or conjugatedmiRNA compounds may be tested using these methods. Furthermore, organssuch as the liver, spleen, heart, lungs and kidney may be investigatedfor signs of modulated viral propagation and/or replication. Animals maybe administered test antiviral compounds by any suitable route includinginjection, inhalation (for example via nasal inhalation) or topically.

In addition to providing methods of identifying host cell miRNAmolecules which may be modulated as a means of modulating (for exampleinhibiting) viral propagation and/or replication, the present inventionprovides specific host cell miRNA molecules which may be targeted formodulation to inhibit viral propagation and/or replication in hostcells. These host cell miRNA molecules may be identified by the methodsdescribed in the first aspect of this invention. It should be understoodthat host cell miRNAs may have sequences which are highly conservedacross a number of different species. As such, human homologues ororthologues of miRNA molecules identified in other animals, for examplerodents etc, may exist. Using the sequence of the miRNA moleculesidentified in cells of certain animal species, it may be possible toprobe for identical or homologous sequences in, for example human cells.Homologous sequences may possess conserved seed sites.

A number of exemplary host miRNA molecules are listed below and havebeen designated SEQ ID NOS: 1-26 respectively. It should be noted thateach the miRNA molecules provided by SEQ ID NOS: 1-26 is conservedbetween mouse and human cells.

Mir-16 UAGCAGCACGUAAAUAUUGGCG (SEQ ID NO: 1) Mir-30a-3pCUUUCAGUCGGAUGUUUGCAGC (SEQ ID NO: 2) Mir-28 AAGGAGCUCACAGUCUAUUGAG(SEQ ID NO: 3) Mir-128a UCACAGUGAACCGGUCUCUUU (SEQ ID NO: 4) Mir-129-5pCUUUUUGCGGUCUGGGCUUGC (SEQ ID NO: 5) Mir-345 GCUGACCCCUAGUCCAGUGCUU(SEQ ID NO: 6) Mir-222 AGCUACAUCUGGCUACUGGGU (SEQ ID NO: 7) Mir-223UGUCAGUUUGUCAAAUACCCCA (SEQ ID NO: 8) Mir-155 UUAAUGCUAAUUGUGAUAGGGGU(SEQ ID NO: 9) Mir-27b UUCACAGUGGCUAAGUUCUGC (SEQ ID NO: 10) Mir-103AGCAGCAUUGUACAGGGCUAUGA (SEQ ID NO: 11) Mir-346 UGUCUGCCCGAGUGCCUGCCUCU(SEQ ID NO: 12) Mir-542-5p (C)UCGGGGAUCAUCAUGUCA (SEQ ID NO: 13)Mir-199a* UACAGUAGUCUGCACAUUGG (SEQ ID NO: 14) Mir-24aUGGCUCAGUUCAGCAGGAAC (SEQ ID NO: 15) Mir-124a UAAGGCACGCGGUGAAUGC(SEQ ID NO: 16) Mir-34b UAGGCAGUGUAAUUAGCUGAU (SEQ ID NO: 17) Mir-452UGUUUGCAGAGGAAACUGAG (SEQ ID NO: 18) Mir-214 ACAGCAGGCACAGACAGGCAGU(SEQ ID NO: 19) Mir-107 AGCAGCAUUGUACAGGGCUAUCA (SEQ ID NO: 20) Mir-744UGCGGGGCUAGGGCUAACAGCA (SEQ ID NO: 21) Mir-30a-5p UGUAAACAUCCUCGACUGGA(SEQ ID NO: 22) Mir-30b UGUAAACAUCCUACACUCAG (SEQ ID NO: 23) Mir-30cUGUAAACAUCCUACACUCUCA (SEQ ID NO: 24) Mir-30d UGUAAACAUCCCCGACUGGA(SEQ ID NO: 25) Mir-30e UGUAAACAUCCUUGACUGG (SEQ ID NO: 26)

By over-expressing, up-regulating or mimicking the miRNA moleculesprovided by SEQ ID NOS: 1-21, or inhibiting the miRNA molecules providedby SEQ ID NOS: 22-26, it is possible to inhibit viral propagation and/orreplication in host cells.

It should be understood that unlike miRNA molecules which specificallytarget the propagation and/or replication cycles of single viral speciesonly, the miRNA molecules listed as SEQ ID NOS: 1-26 target host genes,cell systems and/or pathways and, when their expression is modulated,display multi-species (or broad spectrum) antiviral activity. That is tosay, each of the miRNA molecules provided herein are effective ininhibiting the propagation and/or replication of a variety of viralspecies (i.e. two or more species) in host cells.

In one embodiment, the specific host cell miRNA molecules which may betargeted for modulation in order to inhibit host cell viral propagationand/or replication are not host cell miRNA molecules modulated by avirus and/or not modulated in all cell types/tissues at all stages inthe life cycle of a virus. In otherwords, the compounds provided by thisinvention may not target host cell miRNA molecules which are up or downregulated in response to a viral infection. For example the miRNAmolecules known as Mir-17/92, Mir-28, Mir-93, Mir-100, Mir-101, Mir-122,Mir-125b, Mir-130b, Mir-146, Mir-150, Mir-155, Mir-203, Mir-218, Mir-223and Mir-382 may be excluded from the scope of this invention.

Accordingly, a second aspect of this invention provides a multi-speciesantiviral compound capable of modulating the expression, function and/oractivity of one or more host cell miRNA molecules, for treating viralinfections, diseases and/or conditions.

A third aspect of this invention provides a multi-species antiviralcompound capable of modulating the expression, function and/or activityof one or more host cell miRNA molecules for the manufacture of amedicament for treating viral infections, diseases and/or conditions.

A fourth aspect of this invention provides a method of treating asubject suffering from a viral infection, disease and/or condition, saidmethod comprising the steps of administering a pharmaceuticallyeffective amount of a multi-species antiviral compound capable ofmodulating the expression of one or more host cell miRNA molecules.

In one embodiment the one or more host cell miRNA molecules are thoseprovided by SEQ ID NOS: 1-26 above.

One of skill will appreciate that the compounds capable of modulatinghost cell miRNA molecules mentioned in the second, third and fourthaspects of this invention might take the form of the miRNA mimic orinhibitor compounds described above.

As far as the miRNA molecules identified as SEQ ID NOS: 1-21 areconcerned, compounds comprising these sequences may be used to mimic orover-express the corresponding miRNA molecules in host cells.Accordingly, a further aspect of this invention provides (a)multi-species antiviral compounds selected from the group consisting ofSEQ ID NOS: 1-21 for use in treating viral infections; (b) the use ofmulti-species antiviral compounds selected from the group consisting ofSEQ ID NOS:1-21 for the manufacture of a medicament for treating viralinfections; and (c) a method of treating viral infections, said methodcomprising the steps of administering a pharmaceutically effectiveamount of a composition comprising multi-species antiviral compoundsselected from the group consisting of SEQ ID NOS: 1-21.

It should be understood that the compositions, medicaments and methodsprovided by this invention may comprise or use one or more of thesequences provided as SEQ ID NOS: 1-21 above. For example, a compositionor medicament for treating a viral infection may comprise two or more ofthe miRNA sequences described herein. Compositions, medicaments andmethods which pool or combine compounds selected from those consistingof SEQ ID NOS: 1-21, may be particularly useful when treating patientsinfected with two or more viruses of different species. Furthermore, thecompositions, medicaments and/or methods described herein may becombined with any number of existing antiviral compounds or treatments.

In one embodiment, the miRNA molecules provided by this invention maycomprise the seed sequence of a miRNA molecule identified as beingpotentially useful for treating viral infections and/or diseases and/orconditions caused or contributed to by one or more viral species. Forexample, a seed sequence may comprise the first 1-8 or 2-7 nucleotidesof the 5′ end of a miRNA molecule, including those listed above as SEQID NOS 1-26. One of skill in this field will appreciate that since theseed sequence is generally the functional part of a miRNA molecule, theremainder of the miRNA sequence may be highly variable. In particular,one embodiment of this invention relates to miRNA molecules comprisingthe seed sequences of the miRNA sequences designated SEQ ID NOS: 10, 11and 20 above.

In a sixth aspect, the present invention provides pharmaceuticalcompositions comprising any of the compounds described above (forexample, the host cell miRNA modulating (mimic and/or inhibitor)compounds including SEQ ID NOS: 1-26), in association with apharmaceutically acceptable excipient, carrier or diluent. Suchcompositions may find application in, for example, the treatment ofviral infections and/or diseases and/or conditions caused or contributedto by, viruses.

Preferably, the pharmaceutical compositions provided by this inventionare formulated as sterile pharmaceutical compositions. Suitableexcipients, carriers or diluents may include, for example, water,saline, phosphate buffered saline, dextrose, glycerol, ethanol, ionexchangers, alumina, aluminium stearate, lecithin, serum proteins, suchas serum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water salts or electrolytes, such as protaminesulphate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycon,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polypropylene-block polymers, polyethylene glycol and woolfat and the like, or combinations thereof.

Said pharmaceutical formulation may be formulated, for example, in aform suitable for oral, parenteral or topical administration.Pharmaceutical compositions formulated for topical administration may bepresented as an ointment, solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water liquid emulsion.

One of skill will appreciate that the host cell miRNA modulatingcompounds provided by this invention may be formulated foradministration in a variety of different ways. For example, the hostcell miRNA modulating compounds provided by this invention (for examplethose detailed as SEQ ID NOS: 1-21) may be provided in the form of avector for introduction and expression in a cell.

In addition to the above, the present invention provides a means ofidentifying host genes, cell systems or pathways (for example innateimmune pathways involving for example Tol receptors, Tol-Like receptorsand the like) which may be targets for antiviral therapy. For example,by using the methods described in the first aspect of the invention toidentify host cell miRNA molecules which may be modulated to inhibitviral propagation and/or replication, it may be possible to identify thespecific genes, cell systems and/or pathways targeted by the host cellmiRNA. Identification of the genes, cell systems and/or pathwaysassociated with, or modulated by the host cell miRNA moleculesidentified by a method according to the first aspect of this invention,may provide further targets for antiviral therapies. By way of example,the inventors have determined that genes modulated by mir-30, includethose listed below:

-   -   (1) ankyrin repeat, family A (RFXANK-like), 2    -   (2) isocitrate dehydrogenase 1 (NADP+), soluble    -   (3) kelch-like 20 (Drosophila)    -   (4) LIM homeobox protein 8    -   (5) transcription factor Dp 1    -   (6) collagen triple helix repeat containing 1    -   (7) glucosamine-6-phosphate deaminase 1    -   (8) dpy-19-like 1 (C. elegans)    -   (9) neuron specific gene family member 1    -   (10) transmembrane protein with EGF-like and two        follistatin-like domains 1    -   (11) twinfilin, actin-binding protein, homolog 1 (Drosophila)    -   (12) PRKC, apoptosis, WT1, regulator    -   (13) SET domain containing (lysine methyltransferase) 7    -   (14) cDNA sequence BC031353    -   (15) like-glycosyltransferase    -   (16) LIM and calponin homology domains 1    -   (17) scavenger receptor class A, member 5 (putative)    -   (18) unc-5 homolog C (C. elegans)    -   (19) zinc finger, DHHC domain containing 17    -   (20) c-abl oncogene 1, receptor tyrosine kinase    -   (21) phosphatidylinositol transfer protein, membrane-associated        2    -   (22) retinoic acid receptor, gamma    -   (23) LIM homeobox protein 9    -   (24) N-acetylglucosamine-1-phosphodiester        alpha-N-acetylglucosaminidase    -   (25) procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline        4-hydroxylase), alpha II polypeptide    -   (26) sarcoglycan, beta (dystrophin-associated glycoprotein)    -   (27) SNAP-associated protein    -   (28) UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase,        polypeptide 6    -   (29) dapper homolog 1, antagonist of beta-catenin (xenopus)    -   (30) phosphatidylinositol-5-phosphate 4-kinase, type II, alpha    -   (31) protein tyrosine phosphatase, non-receptor type 13    -   (32) ring finger protein 122    -   (33) sodium channel, voltage-gated, type II, alpha 1    -   (34) WD repeat domain 7

In addition, the genes/pathways regulated by miR-199a are detailed inFIG. 16.

In view of the above, it is apparent that ephrin receptor signalling,thrombin signalling, inositol phsophate metabolism and cytoskeletonorganisation are important pathways modulating broad pro- or anti-viralcellular processes.

One of skill will appreciate that while the present invention has beendescribed with reference to viral infections, diseases and orconditions, the methods, medicaments and compositions described hereinmay also find application in the treatment of infections diseases and/orconditions caused or contributed to by other pathogens. For example, thescope of this invention may extend to the treatment of bacterial(particularly intracellular bacterial) infections and/or parasiticinfection including those caused or contributed to by protozoanparasites (for example Trypanosomes and the like). In addition, thepresent invention may provide methods of screening for, or identifying,host miRNA molecules which may be modulated to inhibit the replication,propagation and/or intracellular entry of other pathogens such asbacterial and/or protozoan pathogens.

DETAILED DESCRIPTION

The present invention will now be described in more detail withreference to the following Figures which show:

FIG. 1: Agonist-antagonist miRNA screening. (A) Overview of screeningprotocol: miRNA mimics or inhibitors were reverse transfected intoNIH-3T3 cells (6 technical replicates) and incubated for 60 hrs prior tocell viability analysis (n=3) or infection with GFP virus (n=3). (B)Fluorescent growth curve of GFP-reporter viruses in NIH-3T3 cells; Y1axis shows values for MCMV and MHV-68 and Y2 shows values for HSV-1;error bars depict standard deviation of 3 technical replicates. Whitelines indicates time points used for subsequent analysis. (C) Normalizedfluorescence data (at 70 hours post infection, hpi, for MCMV and 57 hpifor MHV-68 and HSV-2). Negative controls (RISC-free siRNA, C. elegansmiR-mimic or C. elegans miR-inhibitor) have <10% effect on GFP signalcompared to >50% knock-down using siRNA targeting GFP (n=3, error barsdepict standard deviation).

FIG. 2: Viral growth curve of MCMV in NIH-3T3 cells (the virus isengineered to encode a GFP protein used for detection with a polar starplate reader). The GFP signal correlates with viral accumulation inthese cells over time. The goal in developing the assay was to be ableto examine the effects of over expressing or inhibiting microRNAs onviral replication in a semi-high throughput way. The model is that an“anti-viral” miRNA will inhibit replication when over expressed(compared to a neutral control), and conversely, a miRNA that normallyinhibits replication will cause an increase in replication when it isinhibited. The neutral controls used are C. elegans microRNAs notencoded by the mouse that should not target mouse genes and are notexpected to have an effect on replication. This figure shows two“hits”—one that decreases replication and one that increasesreplication. Although we take measurement for ˜6 days, the standarddeviation is higher at longer time points and we therefore analyse thedata around ˜70 hrs, where the virus is in the linear phase of growth.

FIG. 3: Example of the growth curves obtained with the other 2 virusesused, MHV and HSV-1. Since the growth kinetics are different than MCMV,we analyze data at 58 hours, where the virus is in the linear phase ofgrowth and the signal to background is high. These curves show theneutral controls and a positive control (siRNA against GFP).

FIG. 4: Shown in opals are hits that have >1.5 or <1.5 effect onreplication. The microRNAs in the lower left quadrant are consideredcandidates for “anti-viral” microRNAs that could be mimicked withsynthetic reagents as a therapeutic strategy. One miRNA family, mir-30(mir-30a-5p, mir-30b, mir-30c, mir-30d, mir-30e) resulted in >1.5×increase in replication compared to controls in all three viruses (datashown for MCMV above, these are in the upper right quadrant).

FIG. 5: Shows the GFP signal obtained for all 3 viruses using the mimiclibraries (x axis is the different miRNA mimics, y axis is the GFPsignal reflecting extent of replication). As is shown, there are severalmicroRNAs that result in decreased replication in all three viruses whenover-expressed. There are other microRNAs (mir-30 family) that result inincreased replication when over-expressed. We have made the cutoffs asdescribed in FIG. 5.

FIG. 6: Shows some of the “hits” in MCMV. In the left column is thenormalized fluorescent value of the controls—reflecting the amount ofviral replication in cells transfected with the C. elegans microRNAmimic (black bars) or inhibitor (gray bars). The shaded boxes show twostandard deviations of the controls, so values below or above theseboxes are considered to be “hits”. As shown, some miRNAs significantlyinfluence replication when they are over-expressed, “mimic hits”—somedecrease replication and some increase replication. Other miRNAssignificantly influence replication when they are inhibited—“inhibitorhits”. It is the “double hits” are miRNAs that decrease replication whenthey are over-expressed and increase replication when they areinhibited. Having these two corroborating results helps validate/screenthe data for REAL hits. These double hits would be miRNAs that could beused as anti-viral drugs (over-expressing miRNAs in vivo tocontrol/treat an infection). However, not all miRNAs are expressed inthis cell type, therefore, in the inhibitor screen there could be falsenegatives (if a miRNA is not expressed then inhibiting it should have noeffect). Therefore, we rely primarily on mimic data for identificationof the anti-viral microRNA candidates In addition, there are some miRNAsthat seem to be “pro-viral”, in that replication increases in thepresence of the mimic and/or decreases in the presence of the inhibitor.These would be another type of drug target—where inhibiting thismicroRNA would be a method for controlling the infection.

FIG. 7: Representation of broad spectrum anti-viral miRNAs. The samemethods and cut-offs as described for FIG. 5 were used for 3 differentviruses—MCMV, MHV-68 and HSV. A list of 21 mimics that had the sameeffect (down regulation) on all three viruses using these criteria wasobtained. 1 family of microRNAs, mir-30, resulted in >1.5 fold upregulation of replication in all three viruses.

FIG. 8: Initial data with C127 cells and MCMV. Viral growth was measuredas described above, but data analyzed at 51 hours based on the fastergrowth kinetics under these conditions (signal/noise required MOI of0.5).

FIG. 9: Preliminary results in MRC-5 human fibroblasts infected withHCMV at an MOI of 0.5. Fluorescence was measured at 58 hrs postinfection.

FIG. 10: Preliminary results in NIH-3T3 cells infected with SFV withluciferase reporter at an MOI of 0.5. Luciferase was measured at 10 hrspost infection.

FIG. 11: NIH-3T3 cells were reverse transfected with 25 nm mir-30inhibitor or mir-30 mimic for 48 hours prior to infection with MCMV(with GFP reporter). A) Infected cells were visualized 24 hourspost-infection. B). RNA was harvested from cells transfected with mimicor inhibitor for 48 hours and rt-per performed to quantify (relative)expression of mir-30d under these conditions, as well as the othermir-30 family member (mir-30) or a control host microRNA (mir-16) thatshouldn't be effected by the transfection. This shows over-expression(˜40 fold) of mir-30d by mimic and inhibition (˜85%) by inhibitor.

FIG. 12 (A) Validation of anti- and pro-viral effects of miRNAs in humanMRC-5 cells using HCMV. (B) Examination of anti- or pro-viral effects ofmiRNAs on Semiliki forest virus using a replicon expressing renilla. Inall experiments n=4 and error bars indicate standard deviation.

FIG. 13: Changes in miRNA expression in response to MCMV or HCMV: (A&B)Northern blot analysis of total RNA isolated from mouse fibroblastNIH-3T3 cells infected with MCMV at MOI=3 (harvested 24 hrs postinfection) or human fibroblast MRC-5 cells infected with HCMV at MOI=3(harvested 48 hours post infection). C

FIG. 14: Validation of anti- and pro-viral properties of miRNAs(individual and pooled) in both mouse and human cells. C: COX-2 isdown-regulated upon miR-199a-3p over-expression and is upregulated uponmiR-199a-3p inhibition in both mouse and human cells

FIG. 15: Genes regulated by miR-199a. Accordingly, miRNAs can act asantivirals-regulating multiple networks, that are important/required forviral life cycles, simultaneously.

MATERIALS & METHODS

Cells & Viruses

The viruses used in this study are described elsewhere. Briefly,mcmv-gfp encodes a gfp expression cassette in front of the mcmv ie2 gene[1], a gene that is under immediate early control and is non-essentialfor growth in tissue culture [2]. Wild-type and mcmv-gfp viruses werepropagated in NIH-3T3s and titred in p53 MEFs as described elsewhere[3]. The MHV-gfp virus is officially termed “LHΔgfp”, and contains aninsertion cassette inserted in the 5′end of the genome that encodes gfpdriven by the human cytomegalovirus immediate early promoter. Thiscassette replaces nt 1-3223, which encodes viral factors m1 and vtRNAs(the deletion of which does not significantly alter viral growthkinetics in vitro, B. Dutia, unpublished data) [4]. The LHΔgfp viralstocks were prepared on BHK-21 cells as described elsewhere [5]. TheHSV-gfp27 virus encodes a gfp cassette that replaces the ICP27 gene andis under control of the natural ICP27 promoter [6].

Transfection & Screening Protocol

MicroRNA mimics or inhibitors were reverse transfected into NIH-3T3cells at a final concentration of 25 nM in 0.4% Dharmafect 1(transfection reagent), with 1.5×10^4 cells per 96 well. Lipid wasdiluted to 4% in 10 uL of serum free media (optimem) for 5 minutes priorto mixing with miRNA mimics; the mimics+lipid (10 uL+10 uL) were thenincubated 20 minutes in serum-free media (optimem) prior to addition ofcells (80 uL at 1.875×10^4/ml) in Dulbecco's modified Eagle's medium(DMEM; Invitrogen) supplemented with 10% calf serum. Transfected cellswere then incubated 60 hrs at 37 C, 5% CO2, after which the media wasremoved and cells were infected with virus at an MOI of 0.2. Virusesused were murine cytomegalovirus (BAC-derived strain with GFP insertedunder immediate early control), mouse gammaherpesvirus (BAC-derivedstrain with GFP reporter) or herpes simplex virus-1 (BAC-derived strainwith GFP reporter) or Semliki Forest virus (luciferase reporter) in 2%Calf serum, DMEM media+1% penicillin-streptomycin. Cells were inoculatedwith the virus for 1 hr at 37 C, 5% CO2, after which virus was removedby flicking the plate, followed by addition of 100 uL phenol-red freeDMEM media+1% penicillin-streptomycin. Gas-permeable membranes were thenaffixed to the plate to prevent evaporation and plates were incubated at37 C, 5% CO2 for up to 140 hours. The fluorescent signal was measuredevery in the linear growth time range (50-80 hrs) for MCMV, MGHV, andHSV-1 infected cells using a fluorescent plate reader. For infectionswith Semliki Forest Virus, luciferase was measured between 2-10 hrs postinfection. For validation studies, microRNA mimics or inhibitors werereverse transfected into C127 cells at a final concentration of 25 nM inLipofectamine 2000 at 0.3% (transfection reagent), with 1.5×10^4 cellsper 96 well, maintained in 10% Calf serum, DMEM media+1%penicillin-streptomycin prior to infection with MCMV (as above).MicroRNA mimics or inhibitors were reverse transfected into MRC-5 cellsat a final concentration of 25 nM in Dharmafect 1 at 0.3% (transfectionreagent), with 1.5×10^4 cells per 96 well. Transfected cells were thenincubated 60 hrs at 37 C, 5% CO2, after which the media was removed andcells were infected with human cytomegalovirus (GFP reporter) at an MOIof 0.5. The fluorescent signal was measured in the linear growth rangeunder these conditions (40-70 hr postinfection).

Cell Viability Assays

Following reverse transcription and mock-infection, the effect of mousemiRNAs on cell viability was assessed with Cell titre blue assay(Promega), using a cut-off of 80%. Some miRNA mimics impacted theability of NIH-3t3 cells to adhere properly during reverse transfectionand this could also be identified by cell titre blue (since cells werewashed away in the protocol) and confirmed by visual inspection. MiRNAsthat were toxic or interfered with adherence to the plates (26 of the301 examined) were removed from further analysis. miRNA mimics andinhibitors identified as “hits” in the viral assays were re-examined forimpact on viability as above.

Data Normalization and Analysis for Screening

For each virus, the miRNA mimic and inhibitor libraries were screened intwo independent experiments, each with a minimum of 3 technicalreplicates.

For normalization, the background signal from uninfected cells wassubtracted from the corresponding foreground signal for each well. Datawere then transformed to log 2 scale. Variation in fluorescent intensitybetween individual plates within a given screen was normalised to themedian of control wells included on each plate: non-transfected cells,cells transfected with RISC-free siRNA, C. elegans miR-67 mimic and C.elegans miR-67 inhibitor. A positive control for transfection efficiency(gfp siRNA) was included in each screen, requiring knockdown of >50%.

Influenza Virus Plaque Assay

MDCK cells were reverse transfected with 25 nm miRNAs at 1×10^6 cellsper well in a 6 well plate using DharmaFECT 3_at 0.3% (Lipid). 48 hrspost transfection cells were infected with Mouse adapted Influenza virus(A/WSN/33) and incubated for 1 hr. After infection 2 ml of media wasadded with 2% agarose and 0.1 mg/ml of N-acetyl trypsin. Infected cellswere then incubated at 37 c and 5% Co2 for 3 days. Cells were then fixedwith 10% neutral buffered formalin for 24 hrs. Plaques were countedafter brief staining in 0.1% toluidene blue.

Hep2 Cells/HSV Experiment

Hep2 cells were reverse transfected with 25 nm of miRNAs at 1.5×10^4cells/well in a 96 well plate using DhannaFECT 3_at 0.3% (Lipid). 48 hrspost transfection cells were infected with HSV-1 (GFP virus) at moi of0.5. The fluorescence signal was measured over time.

Pooling Experiments:

The concentration of individual miRNA was adjusted based on the numberof miRNAs in a pool so as to make the final concentration of the miRNApool at 25 nm. miRNA pool were transfected the same way as otherindividual miRNAs. The reason we chose to examine miR-23,24,27 is thatthey all come from the same cluster.

Results

Agonist-Antagonist miRNA Screen.

The agonist-antagonist screening protocol reported here involvesover-expressing or inhibiting miRNAs by transfection of synthetic mimicsor inhibitors, followed by analysis of the impact on viral growth (usingviruses encoding GFP reporters; FIG. 1A). Screening was conducted in amurine fibroblast cell line (NIH-3T3) which supports replication ofrepresentatives of all three herpesviral families: murinecytomegalovirus (MCMV), mouse gammaherpesvirus (MHV-68) and herpessimplex virus-1 (HSV-1), FIG. 1B. Despite the large evolutionarydistance between these viruses is (˜200 million years [19]) all threeviruses require, manipulate and evade common host cell processes (cellcycle, apoptosis, interferon response) which are expected to beregulated by miRNAs and can be interrogated in vitro. Cells wereinfected at a low multiplicity of infection (0.2-0.5), such that thefluorescent signal detected by ˜60-70 hrs is based on multiple rounds ofreplication and the impact of a miRNA on any stage of the replicationcycle should therefore be detectable. One assumption in this screeningapproach is that transfection alone does not itself impact viralreplication. This is confirmed by transfections using C. elegans miRNAmimics or inhibitors or a “RISC-free” siRNA (which gets taken up bycells but not incorporated into RISC). As shown in FIG. 1C, thesereagents have less than a 10% effect on the GFP signal, compared to >50%knockdown with a siRNA directed against GFP. Controls were included ineach plate to normalize plate-to-plate variation in fluorescentintensity. To account for miRNA mimics or inhibitors that result ingeneral toxicity to the cells, viability assays were performed inparallel. Twenty six miRNA mimics (representing 8% of the library) wereexcluded from analysis due to toxicity to cells and/or altered adherenceproperties; none of the inhibitors were scored as toxic.

The Effect of Murine MicroRNAs on the Growth of Murine Cytomegalovirus,Mouse Gammaherpesvirus and Herpes Simplex Virus-1 in Murine FibroblastCells (NIH-3T3).

The effect of murine microRNAs on the growth properties of threedifferent viruses; murine cytomegalovirus, mouse gammaherpesvirus andherpes simplex virus-1 was examined in murine fibroblast cells(NIH-3T3), using genome wide miRNA-mimic libraries. FIG. 2 shows thegrowth curve of MCMV with neutral controls and representative “hits” andFIG. 3 shows the growth curve of MHV and HSV-1 with neutral controls anda positive control (siRNA against the reporter—GFP).

Four miRNAs represent the highest confidence anti-viral microRNAs in allthree herpesviral subfamilies (based on a growth defect when the miRNAis over-expressed and increased quantity of virus when the miRNA isinhibited): mir-199a, miR-214, miR-24 and miR-103. Two miRNAs, miR-30band miR-30d, show the opposite properties (increased growth whenover-expressed and decreased growth when inhibited). Combining this workwith expression analysis, we demonstrate that mir-199a and miR-214(which derive from a common cluster) are down-regulated upon infectionin both murine and human cytomegalovirus. Expression and networkanalysis suggests that mir-199a-3p regulates a range of genes involvedin the immune response, cellular movement and immune cell trafficking.Pooling experiments further demonstrate that miRNAs which are clusteredtogether (miR-199a-5p, mir-199a-3p and miR-214) can provide additiveeffects in inhibiting viral growth capacity. Host miRNAs are a tune-ableand consequential feature of viral infection and provides the firstevidence that these molecules hold broad anti-viral potential againstmultiple viruses.

To define microRNA hits, the normalized fluorescent values obtained forthe screens were plotted with a given virus as shown in FIG. 4. The yaxis represents values obtained in one screen (median values based onn=3) and the x axis represents values obtained in another independentscreen (FIG. 3 shows the 2 independent MCMV screens). We excluded mimicsthat fell outside 2 st-deviations (dashed lines) for the 2 screens asthese are expected to be outliers. We also excluded from this analysismiRNAs that had toxicity or adhesion effects (removed 26 of the total301 unique mouse miRNAs), based on cell-titre blue assay and visualinspection of wells. We found a total of 21 miRNAs that resultedin >1.5× decrease on replication in two independent screens with all 3viruses when transfected into cells prior to infection (listed in Table1 below)—see FIG. 8; and one family of microRNA, mir-30 which resultedin >1.5× increase on replication in all 3 viruses.

In Vitro Screening Results—Inhibitor Libraries

The results from screening microRNA inhibitors may be used tocorroborate results, however, it is important not to use these resultsto “rule out” any of the hits. This is for 2 reasons: 1) not all of themicroRNAs are expressed in the cell type we're using and therefore thereare likely to be a number of false negatives for inhibitor results. 2)the anti-viral effect of microRNAs may require them to be expressed athigher than endogenous levels.

Validation in Other Cell Types

The microRNAs are tested in other cell types to determine whether theyperform the same pro- or anti-viral functions. The screening (asdescribed above) was done in NIH-3t3 (fibroblast) cells and C127(epithelial) cells (see FIG. 7). The results of these screens show thata number of the microRNAs listed as SEQ ID NOS: 1-22 have the same anti-or pro-viral function in the different cell types. This is strongevidence that the miRNA host targets are expressed in the different celltypes.

Validation in Human DNA Viruses

All of the microRNA hits identified in our screen have either aperfectly identical sequence in human, or at a minimum, a conserved seedsite (nt 2-7)—(implying that the targets and function of these microRNAsare conserved in mouse and human).

We have screened a number of the microRNAs listed in Table 1 and 2 andthey have been identified as having the same anti- or pro-viral functionin human cells with HCMV (see FIG. 9).

Validation in RNA Viruses

We have screened a number of the microRNAs listed as SEQ ID NOS: 1-22and they show anti-viral microRNA function against Semliki-forest virusin NIH-3t3 cells.

miRNAs with Conserved Anti- or Pro-Viral Properties in all ThreeHerpesviral Subfamilies

Mouse miRNAs that negatively impact viral growth in this assay aredefined as those that lead to decreased fluorescence in the mimiclibrary and increased fluorescence in the inhibitor library. A commonmetric for qualifying such “hits” is those that falls 1 or 2 standarddeviations outside the mean of negative controls (reviewed in [20]).However, analysis of the mimic and inhibitor data suggests a starkdifference in distributions and magnitude of effects. We therefore optedfor the statistics-based hit selection method, “rank product”, whichdoes not make assumptions about underlying data distributions and isrobust against outlier values [21]. “Hits” are defined as mimics orinhibitors that result in a consistently high or low fluorescent signal(in relation to all of the other mimics or inhibitors) and statisticalweight is based on replication between experiments [21]. There is aclear correlation between the rank product p value and the change influorescence induced by a mimic or inhibitor (compared to negativecontrols). Using the combined datasets for all three viruses (n=19;FDR<0.01) rank product analysis identified 4 high-confidence anti-viralmiRNAs (decreased fluorescence in the mimic library, increasedfluorescence in the inhibitor library): miR-199a-3p, miR-214, miR-103and miR-24 and 3 high-confidence pro-viral miRNAs (increasedfluorescence in the mimic library, decreased fluorescence in theinhibitor library)—mir-30b, mir-30d and mir-93. With the exception ofmir-93, all of these miRNAs validated in subsequent analysis (FIG. 15).Analysis of individual datasets revealed differences between viruses(e.g. mir-29b is strongly anti-viral in HSV-1 but not MCMV or MHV-68 andmiR-378 is pro-viral in MCMV but not MHV-68 or HSV-1). To equate thechange in fluorescent signal of these miRNA mimics and inhibitors withthe change in quantity of infectious virus, standard plaque assays wereperformed with the wild-type MCMV virus. At 70 hours post infection themimics result in a ˜log-fold effect on quantity of infectious viruswhereas inhibitors result in ˜2 fold changes. Growth curve analysisfurther demonstrates 1-2 log fold decrease in infectious virus based onmiR-199a-3p and miR-214 mimics and a ˜5 fold increase in quantity ofvirus with the miR-30 mimic. Notably, these high confidence hits areperfectly conserved in mouse and human and the same anti- and pro-viralproperties are observed when examining the human CMV virus in humancells (FIG. 15). To gain perspective on the breadth of these anti- orpro-viral effects, we also examined Semiliki forest virus, an alphavirusthat is evolutionary unrelated, using a replicon system which replicatesin NIH-3T3 cells. As shown in FIG. 13D, as with the herpesviruses,mir-199a-3p and miR-214 display anti-viral properties against SFVwhereas miR-30 shows pro-viral properties.

Regulation of Host Signalling Networks by mir-199a-3p.

Given the large number of potential targets of any given miRNA (mostrecent estimates at ˜300), it may not be that one specific target (oreven a handful of targets) sufficiently explains a miRNA-basedphenotype. For example, one of the previously reported targets ofmiR-199a-3p is the prostaglandin synthesis COX-2. Inhibition of thisgene has already been shown as an anti-viral strategy in multipleherpesviruses and could potentially explain the anti-viral properties ofmiR-199a-3p. Consistent with this, COX-2 is down-regulated uponmiR-199a-3p over-expression and is upregulated upon miR-199a-3pinhibition in both mouse and human cells (FIG. 14C). However, COX-2 isreported to be targeted by several other miRNAs (e.g. miR-16, xx), whichdo not display the same anti-viral properties. To obtain an unbiased(and more holistic) view of the gene networks that might contribute tomiR-199a-3p function, global transcription analysis was carried out withboth over-expression and inhibition. FIG. 15 shows a list of the mostsignificant genes and networks regulated by mir-199a-3p. Several ofthese are important to a number of viruses and have been shown torepresent drug targets individually.

TABLE 1 Summary of data MCMV MHV HSV-1 SFV HCMV HSV-1 INFLUENZA PrioritymiRNA 3T3 3T3 3T3 3T3 MRC-5 HEP2 MDCK  1 miR- ✓ ✓ ✓ ✓ ✓ ✓ — 199a-3p  2miR-214 ✓ ✓ — ✓ ✓ ✓ n/a  3 miR-346 ✓ ✓ ✓ ✓ — ✓ n/a  4 miR-542 ✓ ✓ ✓ ✓ ✓✓ ✓  5 miR-744 ✓ ✓ ✓ ✓ ✓ ✓ n/a  6 miR-24 ✓ ✓ ✓ — ✓ ✓ n/a  7 miR-103 ✓ ✓✓ — ✓ ✓ n/a  8 (but mir-30 ✓ ✓ ✓ ✓ ✓ — n/a multiple) family inhibitors 9 miR-452 ✓ ✓ ✓ ✓ ✓ ✓ n/a 10 miR-27b ✓ ✓ ✓ — ✓ — n/a 11 miR-155 ✓ ✓ ✓ ✓n/a ✓ n/a 12 miR-222 ✓ ✓ ✓ — n/a ✓ n/a 13 miR-223 ✓ ✓ ✓ ✓ n/a ✓ n/a 14miR-345 ✓ ✓ ✓ ✓ n/a ✓ n/a 15 miR-28 ✓ ✓ ✓ ✓ n/a — n/a 16 miR-107 ✓ ✓ ✓n/a n/a n/a n/a 17 miR-124a ✓ ✓ ✓ ✓ ✓ n/a 18 miR-128 ✓ ✓ ✓ — n/a ✓ n/a19 miR- ✓ ✓ ✓ — n/a ✓ n/a 129-5p 20 miR-30a- ✓ ✓ ✓ ✓ n/a ✓ n/a 3p 21miR-34b ✓ ✓ ✓ ✓ ✓ ✓ n/a 22 miR-16 ✓ ✓ ✓ ✓ ✓ ✓ n/a Pool 1 199a- ✓ n/a n/an/a ✓ ✓ n/a 3p/199a- 5p/214 mimic pool Pool 2 23/24/27 ✓ n/a n/a n/a n/an/a n/a mimic pool

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The invention claimed is:
 1. A method of treating a subject having oneor more viral infections, viral diseases and/or viral conditions, saidmethod comprising the steps of administering a pharmaceuticallyeffective amount of a multi-species antiviral microRNA (miRNA) capableof modulating or mimicking the expression, function and/or activity of ahost cell miRNA molecule, wherein the host cell miRNA is miR-199a havingthe nucleotide sequence of SEQ ID NO:14.
 2. The method of claim 1,wherein the multi-species antiviral miRNA is effective against three ormore viral species.